I realize that this reply is a bit late but our E-mail port was not working
for a few days and I ended up getting this back a few days later :-D.G.
-------Lenny writes----------------------------------------------------------
>Howdy Net-Folk
> Got a small cloning problem. I'm trying to construct a plant
>promoter testing T-DNA vector. I have a binary T-DNA with a GUS reporter
>gene/NOS terminator with no promoter and a single HindIII site to drop
>in a putative test promoter. I constructed it for my thesis work. Now
>I want to test some other putative promoter fragments that have internal
>HindIII sites, so I deleted some miscelenious restriction sites around the
>T-DNA vector and want to add the pBLUESCRIPT MCS. I have deleted the HindIII
>site from pBLUESCRIPT-II-SK+ and saved this new plasmid. Next I cut out
>the MCS with BssHII, flushed with T4-Pol, ligated on HindIII linkers, cut
>with HindIII, and tried to ligate into the HindIII site in the T-DNA. I
>don't get any possative clones.
>Questions:
>1. Does anyone know any peculiar problems with cloning poly-linkers?
>2. Does anyone know any tricks to doing the above?
------------------------------------------------------------------------------
Hi Lenny,
1. In my experience cloning a MCS is fairly easy,
in fact one should takeprecautions
to get rid of oligos of the MCS when
doing double cuts within it. ( You can
PEG theplasmid DNA away form the oligomer).
2. What I would do in your situation is blunt end
the MCS into the HindIII site.
Here's a trick you can use to clone
any piece of DNA (with the correct ends)
into a vector by blunt end ligation.
This simplifies many constructs and
allow you to regenerate restriction sites
if you need to by selecting to cut with an
enzyme that, after Klenow treatment,
will leave the correct base.
Take your DNA fragment ( HindIII digested)
containing your E.coli origin ( Your
T-DNA vector) and treat with CIP.
If the size is about that of pUC or pBR322
you will needabout 200ng for your ligation
in a 20uL reaction. I would cut &cip 5 ug and
use the appropriate volume.
Prep your MCS fragment, if you can use
enzyme sites that can beblunted with Klenow
treatment (ie the 3' recessed end; which BssHII
qualifies) I much prefer Klenow over T4
polymerase just because I've used it more
successfully. You want as much as you can
getof this fragment so I would cut 5-10 ug of
plasmid. Purify the MCS, a small DNA
fragment like this you may want to go to
acrylamide gels.
Klenow treatment:
Mix 200 ng of your CIP treated vector (1-2 uL)
with;
2uL of 10X nick translation buffer (containing MgCl2 instead of
he sulfate: this is important as you will be doing the ligation
in this reaction and ligase (T4 DNA ligase) does not seem to
work in NTB with Mg(SO4)2; 100 uM dNTPs; then
add your MCS fragment ( about 1/2 your yeild or 5 ugs of plasmid
worth) and H20 to make 20 uL total. Add about 2.5 units of good
Klenow ( stuff that hasn't been pulled in and
out of the freezer too much) and incubate on
the bench (RT) for 20 min.
Then I add 1/2 uL of 50 mM ATP
1/2 uL of 250 mM DTT
H2O to 25 uL and then add
5 to 8 or so Units of Good T4 DNA ligase (1 uL)
(I use Boehringer Mannheim High concentration)
mix and incubate O/N
at 10 - 12 degrees C. ( This is Imp as this
increases
the half life of ligase, I've read) Also the high concentrations
of Ligase are one of the most
IMP things to getting a blunt
end ligation to work. Others
are concentration of ends (plasmid
and fragment) so I keep them
to 200 ngs in 20 uL for
plasmids of pUC and pBR322
sizes and then I load in the
fragment to be cloned ( In
your case the MCS, use about the
amount you get from 5 to 10
ugs of an average sized plasmid)
Finally, make sure your ATP is
good! Usually this is the problem
I find when I'm trouble
shooting others ligation woes.
The next morn, dilute 1:1 (or for
the purist, add Na Acetate and EtOH pptt,
dissolve in H2O)and electroporate 2ul of 20
into your favourite E.coli strain. I have made
many plamids this way some of which are listed
in Gietz and Sugino (1988) Gene 74: p527-534. If
your vector is Cippedwell you should be able to
get your clones within your first 20 minis.
If I can't find what I want
in 20 I redo the ligation modifying
the concentration of ends, usually
increasing the amount of
fragment added and RE CIPPing the vector.
Once you've got some clones the
only thing you have
to worry about is the orientation of the
fragment in your vector.
Good Luck on your cloning.
I would be happy to answer any other specifics
about this technique.
Dan Gietz
University of Manitoba
GIETZ at BLDGHSC.LAN1.UMANITOBA.CA